Here, we describe a high resolution whole-mount imaging method in the entire adult mouse ear skin, which enables us to visualize branching morphogenesis and patterning of peripheral nerves and blood vessels, as well as immune cell distribution.
Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching morphogenesis and patterning, as well as immune cell distribution at a cellular level. The analysis of peripheral nerve and blood vessel anatomical structures in adult tissues provides some insights into the understanding of functional neuro-vascular wiring and neuro-vascular degeneration in pathological conditions such as wound healing. As a highly informative model system, we have focused our studies on adult ear skin, which is readily accessible for dissection. Our simple and reproducible protocol provides an accurate depiction of the cellular components in the entire skin, such as peripheral nerves (sensory axons, sympathetic axons, and Schwann cells), blood vessels (endothelial cells and vascular smooth muscle cells), and inflammatory cells. We believe this protocol will pave the way to investigate morphological abnormalities in peripheral nerves and blood vessels as well as the inflammation in the adult ear skin under different pathological conditions.
Skin is comprised of three layers: the epidermis, dermis and hypodermis. It has been used as a model system to study stem cell maintenance, differentiation, and morphogenesis in development as well as the regeneration, tumorigenesis, and inflammation in adult. Skin is richly vascularized and innervated such that the development of the peripheral nervous system and vascular system is well-coordinated.
We have previously demonstrated a whole-mount embryonic skin imaging technique with multiple labeling to study intact peripheral nerves and blood vessels including their cellular components1,2,3,4: sensory axons, sympathetic axons, Schwann cells in nerves, endothelial cells, pericytes, and vascular smooth muscle cells (VSMCs) in blood vessels. During the angiogenesis, a primary capillary network undergoes intensive vascular remodeling and develops into a hierarchical vascular branching network. In the developing dermis/hypodermis, arteries branch alongside peripheral sensory nerves and veins then form adjacent to the arteries. After the hierarchical vascular network is thoroughly covered with VSMCs, sympathetic nerves extend along and innervate large-diameter blood vessels1,5,6. Despite the significance in the close association between the developing nervous and vascular systems, a major question has been to address what happens to the neuro-vascular networks in various pathological situations in adults. A three-dimensional high-resolution imaging is necessary to appreciate the pathogenesis, along with anatomically recognizable branching morphogenesis and patterning.
Neuronal and vascular morphogenesis in adult mouse skin is commonly analyzed by tissue section staining. Other studies have used whole-mount imaging of the skin to visualize the peripheral nerves and blood vessels, in addition to the hair follicles, sebaceous glands, and arrector pili muscles7,8,9. However, the thickness of adult skin has made it difficult to analyze the skin over its entire depth.
In the present study, we developed a novel high-resolution whole-mount imaging of adult ear skin to overcome these challenges. Ear skin is readily accessible for dissection and subsequent whole-mount imaging of the skin over its entire depth. Thus, it is a straightforward and highly reproducible method that can be applied to compare three-dimensional architecture of peripheral nervous and vascular systems in the skin, with comprehensive quantification measurements. We demonstrated that the alignment of peripheral sensory and sympathetic nerves with large-diameter blood vessels is preserved in the adult skin. The goal of this protocol is to visualize branching morphogenesis, and the patterning of peripheral nerves and blood vessels, as well as the immune cell distribution at a cellular level in adult mouse models in various conditions such as inflammation and regeneration.
All experiments in this section were performed under approval from the National Heart, Lung, and Blood Institute (NHLBI) Animal Care and Use Committee.
1. Adult Mouse Ear Skin Collection
2. Whole-mount Immunohistochemical Staining of Mouse Ear Skin
NOTE: All experiments in the following sections were performed in accordance with the NIH laboratory safety guidelines.
3. Mounting the Ear Skin on Slide
4. Confocal Microscopy
Adult mouse posterior ear skin (Figure 1A) and anterior ear skin (Figure 1B) were immunostained with antibodies to αSMA (red), Tuj1 (green), and PECAM-1 (blue). Posterior skin was immunostained to study neuro-immune distribution using antibodies to CD11b (red) and MBP (green), together with Tuj1 (blue) (Figure 2A). Distribution of CD11b+ inflammatory cells, including macrophages was detected at a single cellular resolution (Figure 2B).
Figure 1: Alignment ofperipheral nerves and blood vessels in adult ear skin. Whole-mount triple immunofluorescence confocal microscopy of posterior and anterior ear skin with antibodies to αSMA (red), Tuj1 (green), and PECAM-1 (blue) is shown. (A) VSMC-covered large-diameter blood vessels align with peripheral nerves in the posterior ear skin. (B) Smaller-diameter blood vessels covered with VSMCs align with smaller-diameter peripheral nerve bundles in the anterior ear skin. Scale bar = 1 mm Please click here to view a larger version of this figure.
Figure 2: Myelination of peripheral nerves and CD11b+ myeloid cell distribution in adult ear skin. Whole-mount triple immunofluorescence confocal microscopy of posterior ear skin with antibodies to CD11b (red) and MBP (green), together with Tuj1 (blue), is shown. (A) Medium-to-large diameter peripheral nerves are myelinated. (B) Close-up image in (A). CD11b+ inflammatory cells distribute evenly in the posterior ear skin. Scale bar = 1 mm (A), 100 µm (B). Please click here to view a larger version of this figure.
This protocol describes the whole-mount immunonohistochemical imaging of adult ear skin for the analysis of neuro-vascular structures and immune cell distribution. We believe this method has numerous experimental advantages for researchers to study branching morphogenesis and the patterning of peripheral nerves and blood vessels, as well as three-dimensional distribution of skin components including immune cells and hair follicles. The results of the imaging can be quantified using imaging softwares for further quantitative analysis.
Proper preparation of the ear skin is critical for the success of this protocol. First, the ear skin should be carefully dissected soon after euthanizing the mouse. The posterior part of the ear skin should be peeled away from the cartilage. Then, the cartilage should be peeled off from anterior skin before staining. Second, connective tissues, adipose tissues, and hairs should be removed gently and thoroughly before mounting. Due to the existence of peripheral nerves on the surface of the skin, careful removal is required to avoid damaging the nerves. Third, the ear skin should be unfolded with the removal of some thick tissues from the ear skin. Finally, the ear skin should be flat-mounted without air bubbles.
One limitation of this protocol is that ear-tagged ear skin is not appropriate for the analysis as ear tag causes a hole or a scar. Therefore, identification of mice by different methods other than ear tag such as labeling on the tail is necessary in case there are multiple mice to analyze.
The entire ear skin can be scanned by confocal microscopy with a tile scan tool, although a previous reports demonstrated that a region of interest can be imaged with a high resolution10. Interestingly, the whole-mount imaging of the entire ear skin reveals distinct branching morphogenesis and patterning of peripheral nerves and blood vessels between the posterior and anterior skin (Figure 1): the posterior skin has large-diameter nerve bundles (20–50 µm) aligned with remodeled large-diameter blood vessels covered with αSMA+ VSMCs (20–60 µm), while the anterior skin has smaller-diameter nerve bundles (<20 µm) aligned with smaller-diameter but remodeled blood vessels covered with αSMA+ VSMCs (<20 µm).
There are a remarkable number of mouse models11 to elucidate the mechanism of human dermatological diseases such as atopic dermatitis12, psoriasis13, wound healing14, and diabetic neuropathy15. We have applied this protocol to the ear skin of diet-induced obesity mice and type 2 diabetic mice to study diabetic neuropathy16. This protocol can be applied from juvenile to adult mouse skin in various pathological conditions.
The authors have nothing to disclose.
We thank K. Gill for the laboratory management and technical support, J. Hawkins and the staff of National Institutes of Health (NIH) Building 50 animal facility for the assistance with mouse care, and R. Reed and F. Baldrey for administrative assistance. Thanks also to S. Motegi and M. Udey for sharing their ear skin dissection protocol, N. Burns for editorial help, and members of the Laboratory of Stem Cell and Neuro-Vascular Biology for technical help and thoughtful discussion. T. Yamazaki was supported by the Japan Society for the Promotion of Science (JSPS) NIH-KAITOKU. This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute (HL005702-11 to Y.M.)
10 x Phosphate Buffered Saline | KD Medical | RGE-3210 | PBS, without Ca2+/Mg2+ |
Hank’s Balanced Salt Solution | Gibco | 14025-092 | HBSS, with Ca2+/Mg2+ |
16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | PFA, fixative, diluted in PBS |
Triton X-100 | Sigma | X100 | Detergent |
Normal goat serum | Gibco | 16210064 | Component of blocking/washing buffer |
Normal donkey serum | Jackson Immuno research | 017-000-121 | Component of blocking/washing buffer |
Curved fine tweezers | Dumont | RS-5047 | |
Curved tweezers | Integra Miltex Vantage | V918-782, V918-784 | |
Filter Unit 0.45 mm | Thermo Scientific | 157-0045 | For filtration |
1 mL syringe | Coviden | 8881501400 | For filtration |
Syringe filter Unit 0.22 mm | Millex-GV | SLGVR04NL | For filtration |
ProLong Gold | Thermo Scientific | P36934 | Anti-fade mounting medium |
Nail Polish | Electron Microscopy Sciences | 72180 | For sealing |
Dissecting microscope | Leica | MZ95 | |
Confocal microscope | Leica | TCS SP5 | |
Photoshop CC 2017 | Adobe | Graphics editor software | |
Illustrator CC 2017 | Adobe | Graphics editor software | |
Image J | NIH | Image processing software | |
Anti-PECAM-1 (CD31) antibody | Millipore | MAB1398Z | Hamster IgG, vascular endothelial cell marker, 1:300 |
Anti-PECAM-1 (CD31) antibody | BD Pharmingen | 553369 | Rat IgG2a kappa, vascular endothelial cell marker, 1:300 |
Anti-aSMA antibody conjugated with cy-3 | Sigma | C6198 | Mouse IgG2a, vascular smooth muscle cell marker, 1:500 |
Anti-EphB1 antibody | Santa Cruz | sc-9319 | Goat polyclonal, venous endothelial cell marker, 1:100 |
Anti-neuron-specific Class III b-tubulin (Tuj1) | Abcam | AB18207 | Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500 |
Anti-Tuj1 antibody | Covance | MMS-435P | Mouse IgG2a, pan-axonal marker, 1:500 |
Anti-MBP antibody | Abcam | AB40390 | Rabbit polyclonal IgG, myelination marker, 1:200 |
Anti-Tyrosine Hydroxylase antibody | Chemicon | AB152 | Rabbit polyclonal, sympathetic neuron marker, 1:500 |
Anti-Peripherin antibody | Chemicon | AB1530 | Rabbit polyclonal, peripheral neuron marker, 1:1000 |
Anti-CD11b antibody | Bio-Rad | MCA74G | Rat IgG2b, inflammatory cell marker (macrophages), 1:50 |
Anti-CD45 antibody | Thermo Fisher Scientific | 14-0451-85 | Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500 |
Anti-CD3 antibody | Bio-Rad | MCA1477T | Rat IgG1, immune cell marker, 1:100 |
Anti-CD45R (B220) antibody | Thermo Fisher Scientific | 14-0452 | Rat IgG2a kappa, inflammatory cell marker, 1:200 |
Anti-GFP antibody | Thermo Fisher Scientific | A11122 | Rabbit polyclonal, 1:300 |
Anti-GFP antibody | Abcam | Ab13970 | Chicken polyclonal, 1:500 |
Anti-b-gal antibody | Cappel | 55976 | Rabbit polyclonal, 1:5000 |
Anti-RFP antibody | Abcam | Ab62341 | Rabbit polyclonal, 1:300 |
Goat anti-rabbit IgG (H+L) Alexa 488 | Thermo Fisher Scientific | A11034 | Rabbit polyclonal secondary antibody, 1:250 |
Goat anti-hamster IgG (H+L) Alexa 647 | Jackson Immuno research | 127-605-160 | Hamster polyclonal secondary antibody, 1:250 |
Goat anti-rat IgG (H+L) Alexa 594 | Jackson Immuno research | 112-585-167 | Rat polyclonal secondary antibody, 1:250 |
Goat anti-mouse IgG2a Alexa 633 | Thermo Fisher Scientific | A21136 | Mouse IgG2a secondary antibody, 1:250 |